Transcription of plasmid ColEl DNA by RNA polymerase in vitro starts at two sites in a region required for maintenance of the plasmid. Certain transcripts that start at one of the sites can be cleaved by RNase H and then act as primers for DNA replication. Transcription from the other site produces a RNA approximately 108 nucleotides long (RNA I). If purified RNA I is added to the transcription reaction containing RNase H, formation of primer is inhibited. Each RNA I can inhibit primer formation by the plasmid that specifies it but has not effect on primer formation by heterologous templates. Thus, the inhibition of primer formation by RNA I is incompatibility specific. Because RNA I does not inhibit initiation or propagation of transcription or the processing of performed precursors, the step that is sensitive to inhibition is probably formation of the hybrid between the primer precursor and the template. This hybrid is the requied substrate for RNase H.